single quadrupole mass spectrometry

Forensic Science International 157 (2006) 57–70 www.elsevier.com/locate/forsciint

A method for screening for various sedative-hypnotics in serum by liquid chromatography/single quadrupole mass spectrometry Hajime Miyaguchi *, Kenji Kuwayama, Kenji Tsujikawa, Tatsuyuki Kanamori, Yuko T. Iwata, Hiroyuki Inoue, Tohru Kishi National Research Institute of Police Science, 6-3-1 Kashiwanoha, Kashiwa-shi, Chiba 277-0882, Japan Received 4 June 2004; received in revised form 18 February 2005; accepted 7 March 2005 Available online 24 May 2005

Abstract A screening method for the detection of sedative-hypnotics in serum is described. The target drugs, which include practically all the sedative-hypnotics distributed in Japan, consisted of 5 barbiturates, 30 benzodiazepine-related drugs and 11 other sedativehypnotics (i.e., apronalide, bromisovalum, chloral hydrate, triclofos, chlorpromazine, promethazine, diphenhydramine, hydroxyzine, zopiclone, zolpidem and tandospirone). Thirty-nine analytes, selected in terms of the pharmacokinetics of the target drugs, in human serum were screened using a combination of mixed-mode solid-phase extraction and liquid chromatography/ electrospray-ionization single-quadrupole mass spectrometry. The detection limits (non-basic analytes, 1–50 ng/ml; basic analytes, 0.1–5 ng/ml) were sufficient to permit the screening of a single therapeutic administration of a target drug. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Drug screening; Benzodiazepines; Serum; Solid-phase extraction; Liquid chromatography/mass spectrometry (LC/MS)

1. Introduction The use of sedative-hypnotics has been traced to various crimes such as murder, sexual assault and traffic accidents. In addition, these drugs have also been associated with suicide by carbon monoxide poisoning and drug overdose. Therefore, it is important to prepare a screening method for all the sedative-hypnotics distributed in each country for determining whether or not sedative-hypnotics are involved in these cases. Several immunoassay kits are available for screening selected sedative-hypnotics, such as benzodiazepines and barbiturates, but some have a limit of detection that may be insufficient to reveal administration of a therapeutic dose [1]. * Corresponding author. Tel.: +81 4 7135 8001; fax: +81 4 7133 9173. E-mail address: [email protected] (H. Miyaguchi).

Capillary gas chromatography/mass spectrometry (GC/MS) is the most reliable technique for drug identification due to its excellent chromatographic resolution and the availability of library-searchable spectral information using electron ionization. As a result, a number of screening methods for sedative-hypnotics that involve the use of GC/MS have been reported [2,3]. However, some of the sedative-hypnotics have low volatility and require derivatization prior to analysis. An additional ionization method (i.e., negative chemical ionization) may be also required to determine sub-ppb levels of sedative-hypnotics in blood [4]. Liquid chromatography/mass spectrometry (LC/MS) is an alternative technique for drug identification. The high sensitivity and wide applicability of LC/MS has led to dramatic changes in drug analysis and a number of drug screening methods using LC/MS(/MS) have been reported [5–9]. In particular, electrospray ionization provides outstanding sensitivity for the detection of most sedative-hypnotics that

0379-0738/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2005.03.011

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are charged, polar substances. While triple-quadrupole LC/MS permits advanced sensitivity and selectivity, singlequadrupole LC/MS has the merits of low cost, ease of maintenance and adequate selectivity, all of which are suitable for general screening [10]. Fractionating extraction is suitable for electrospray ionization because either the positive or negative mode must be chosen along with the charge of the analytes. Mixed-mode solid-phase extraction, the sorbent of which provides both ion exchange and reversed phase properties, is a simple technique that can be used for fractionation [1,6,11]. Both blood and urine are frequently used in drug testing. In general, the total concentration of a drug and its metabolites in urine is higher than that in blood, but urine would require the hydrolysis of a conjugate followed by analyzing phase I metabolites, standards for which are not always commercially available. Using blood for drug screening would avoid most of the problems mentioned above, but a higher sensitivity would be required in order to detect smaller amounts of compounds. The goal of this study was to establish a screening method for sedative-hypnotics use. The target drugs included all the hypnotics that have been approved by the Japanese government for the treatment of insomnia or sleep disorders as of 2004 and included barbiturates, benzodiazepine-related compounds as well as other drug categories [12,13] (Table 1). In addition, the benzodiazepines for the treatment of anxiety and epilepsy (e.g., diazepam and clonazepam) and some sedatives rather than hypnotics (e.g., hydroxyzine) were also included. Herbal medicines were excluded from the list of targets because of their low potency. The present method was developed on serum samples, and 39 analytes were selected in terms of their pharmacokinetics. A mixed-mode solid-phase extraction and a single-quadrupole LC/MS was used for extraction and detection, respectively.

2.2. Standards and solutions The target drugs and the corresponding analytes are shown in Table 1. An analytical standard of 7-bromo5-(2-fluorophenyl)-3H-1,4-benzodiazepine-2(1H)-one, an active metabolite of haloxazolam (Sankyo code, No. 574; see Fig. 1d), was synthesized from the corresponding benzophenone (prepared by the hydrolysis of haloxazolam) and glycine ethyl ester as described in the literature [14]. Amobarbital (Nippon Shinyaku), flutazolam (Mitsui pharmaceutical), lormetazepam (Wyeth), quazepam (SS pharmaceutical), rilmazafone M-4 (see Fig. 1f, Shionogi), fludiazepam and tandospirone citrate (both Sumitomo pharmaceuticals) were generously donated by corresponding pharmaceutical companies. Bromisovalum, phenobarbital, trichloroacetic acid, brotizolam, clotiazepam, etizolam, nimetazepam, nitrazepam and promethazine were purchased from Wako. Other standard compounds were purchased from Sigma. Stock solutions (10 mg/ml) of nonbasic analytes (A, see Table 1) were prepared in acetonitrile:water (7:3, v/v). Stock solutions (1 mg/ml) of basic compounds (B, see Table 1) were prepared in acetonitrile except for zolpidem (1 mg/ml in acetonitrile:water, 7:3, v/ v) and rilmazafone M-4 (0.5 mg/ml in acetonitrile: water, 7:3, v/v). Working mixtures of analytes were prepared from the respective stock solutions of A and B. All stock solutions and working mixtures were kept at 4 8C and no degradation of the compounds was observed in the working mixture (A, 50 mg/ml; B, 5 mg/ml, in water:acetonitrile, 3:1, v/v) by LC/UV measurements after 2 months of preparation. A 10-mmol/l ammonium acetate solution (pH 6.8), for the mobile phase, was prepared daily by the 500-fold dilution of an ammonium acetate stock solution (5 mol/l, filtrated) with fresh Milli-Q water with no subsequent filtration. 2.3. Instrumentation

2. Experimental 2.1. Reagents Methanol, acetonitrile (both HPLC grade), 25% aqueous ammonia, ammonium acetate and ethenzamide (internal standard) were purchased from Wako (Osaka, Japan). Following human sera (a–f) were used: a (male, pooled), Lot No. 122K0424, PN H1388, Sigma (St. Louis, MO, USA); b (male, blood type AB, pooled), Lot No. 083K0477, PN H1513, Sigma; c (pooled), Lot No. R12393, PN 823201, ICN (Irvine, CA, USA); d (individual, male, narcotics free), Lot No. N57743, Cosmo Bio (Tokyo, Japan); e (individual, male), Lot No. N59708, Cosmo Bio; f (individual, female), Lot No. N59732, Cosmo Bio. Serum a was used for the blank serum unless otherwise described. Water was purified and filtered through a Milli-Q Simpli Lab-UV system from Millipore (Billerica, MA, USA). Other chemicals were of analytical reagent grade.

The LC/MS equipment was composed of a 2690 separation module, a ZQ single-quadrupole mass detector and Mass Lynx software (Waters, Milford, MA, USA). A Waters 996 photodiode array detector was used for UV measurements. The chromatographic conditions were based on a LC/MS screening method developed by Adachi and Takahashi [15]. A Symmetry C18 column (Waters, 150 mm
2.1 mm, particle size 3.5 mm) was used for the separation at 35 8C with an Opti-solv in-line filter (0.5-mm pore, Optimize technologies, OR, USA). The mobile phase, delivered at a flow rate of 0.2 ml/min, was a gradient of methanol (B) in 10 mmol/l ammonium acetate (A): 0–5 min, 10% B; 5–45 min, from 10% to 90% linear gradient of B in A; 45–55 min, 90% B; 55–60 min, from 90% to 10% B; 60–80 min, equilibration of the column with 10% B. Electrospray ionization was performed with a Z-spray module (Waters) with no splitting from the column. Acquisition programs were constructed for each fraction, and the

Table 1 The analytes and acquisition parameters

A A A A A A A A

Amobarbital Apronalide Barbital Bromisovalum Pentobarbital Phenobarbital Secobarbital Trichloroacetic acid (TCA)

B B B B B B B B B

Alprazolam Bromazepam Brotizolam Chlordiazepoxide Chlorpromazine Clonazepam Clotiazepam Delorazepam N-Desmethyldiazepam

B

N-Desmethylfludiazepam

B B B B B B B B B B

Diazepam Diphenhydramine Estazolam Etizolam Fludiazepam Flunitrazepam Flutazolam Haloxazolam No. 574 Hydroxyzine Lorazepam

B B B B B B

Lormetazepam Midazolam Nimetazepam Nitrazepam Promethazine Quazepam

Related sedative/hypnotics

Triclofos, chloral hydrate

Cloxazolam, mexazolam Chlordiazepoxide, clorazepate, diazepam, medazepam, oxazolam, prazepam Ethyl loflazepate, fludiazepam, flurazepam, flutazolam, flutoprazepam, quazepam Medazepam

Haloxazolam Cloxazolam, delorazepam, lormetazepam, mexazolam

Nimetazepam

Molecular formula

Mono-isotopic massa

Monitored ions ( m/z )b

Typical retention time (min)

C11H18N2O3 C9H16N2O2 C8H12N2O3 C6H11BrN2O2 C11H18N2O3 C12H12N2O3 C12H18N2O3 C2HCl3O2

226 184 184 222, 224 226 232 238 162

225 185 183 223, 225, 180, 182 225 231 237 161, 163, 117, 119

32.8 30.2 16.5 25.6 32.8 24.9 34.6 5.3

28–40 28–40 10–20 20–28 28–40 20–28 28–40 2.5–10

C17H13ClN4 C14H10BrN3O C15H10BrClN4S C16H14ClN3O C17H19ClN2S C15H10ClN3O3 C16H15ClN2OS C15H10Cl2N2O C15H11ClN2O

308 315, 317 392, 394 299 318 315 318 304 270

309, 316, 393, 300 319, 316, 319, 305, 271,

35.2 31.3 35.7 37.2 44.5 33.0 40.4 37.0 37.7

34.5–36.25 28–34.5 34.5–36.25 36.25–37.75 39.5–50 28–34.5 39.5–50 36.25–37.75 37.4–38.5

60 50 50 40 45 50 45 60 60

C15H10ClFN2O

288

289

36.0

34.5–36.75

50

C16H13ClN2O C17H21NO C16H11ClN4 C17H15ClN4S C16H12ClFN2O C16H12FN3O3 C19H18ClFN2O3 C15H10BrFN2O C21H27ClN2O2 C15H10Cl2N2O2

284 255 294 342 302 313 376 332, 334 374 320

285, 193 256 295 343 303, 305 314 377 333, 335 375 321

38.5 35.3 34.0 36.2 37.2 33.3 38.0 36.7 43.1 35.3

37.75–39.5 34.5–36.75 28–34.5 34.5–36.75 36.25–37.75 28–34.5 37.4–38.5 36.25–37.75 39.5–50 34.5–36.25

60, 90 20 50 50 60 50 60 60, 40 50 45

C16H12Cl2N2O2 C18H13ClFN3 C16H13N3O3 C15H11N3O3 C17H20N2S C17H11ClF4N2S

334 325 295 281 284 386

335, 337 326 296 282 285 387

36.6 38.5 33.7 32.8 41.8 41.7

36.25–37.75 37.75–39.5 28–34.5 28–34.5 39.5–50 39.5–50

40 60 50 50 30 50

311 318 395 321 318 321 307 273

Acquisition period (min)c

Cone voltage (V)d

30 30 30 30, 60 30 30 30 20, 30

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Analyte

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Type

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60 60 50 60 30 28–34.5 36.25–37.75 34.5–36.75 34.5–36.25 28–34.5 A, non-basic analytes; B, basic analytes. a 1 H, 12C, 14N, 16O, 19F, 32S, 35Cl, 79Br and 81Br are used for calculation. b Fragment ions are underlined. c Acquisition periods must be changed along with the retention times. d The minus sign attached to the cone voltage indicates negative mode. The value used for fragmentation are underlined.

30.5 36.5 35.1 35.1 31.7 373 384 343 308, 309 389 Rilmazafone Rilmazafone M-4 Tandospirone Triazolam Zolpidem Zopiclone B B B B B

C17H10Cl2N4O2 C21H29N5O2 C17H12Cl2N4 C19H21N3O C17H17ClN6O3

372 383 342 307 388

Typical retention time (min) Mono-isotopic massa Molecular formula Related sedative/hypnotics Analyte Type

Table 1 (Continued )

parameters (monitored ions, acquisition timetable and cone voltage) are shown in Table 1. Other conditions were as follows: dwell time, 0.1 s; capillary voltage, 3 kV; extractor, 3 V; rf lens, 0 V; source temperature, 100 8C; desolvation temperature, 300 8C; cone gas (nitrogen) flow, 50 l/h; desolvation gas (nitrogen) flow, 350 l/h. When examining the optimum cone voltage, an analytical standard solution was directly introduced into the mass spectrometer and a series of cone voltages (from 10 V to 70 V, 10 V intervals) was applied to the scan acquisition for each standard analyte. 2.4. Adsorption of the analytes on membrane filters

Monitored ions ( m/z )b

Acquisition period (min)c

Cone voltage (V)d

60

The analytes were divided into six groups in order to avoid peak overlapping in the chromatogram, and 100 ml of the mixture (A, 10 mg/ml; B, 1 mg/ml) in water:methanol (9:1 or 1:9, v/v) was filtered through an Ultrafree MC (a centrifugal filter unit with a 0.45-mm polyvinylidene fluoride (PVDF) microporous membrane, Nihon Millipore, Yonezawa, Japan). Differences in peak areas of analytes after filtration were monitored by LC/UV or LC/MS measurements (n = 2). The monitored wavelength and monitored ions are shown in Table 2. 2.5. Sample preparation and extraction procedure A 0.5-ml volume of human serum (spiked with appropriate amounts of the standard mixtures) was mixed with 0.5 ml of 0.1 mol/l HCl. After brief mixing, the solution was applied to an Oasis MCX mixed-mode solid-phase extraction cartridge (Waters, 1-ml syringe volume, 30-mg sorbent weight) conditioned with 1 ml of acetonitrile and 2 ml of water. The cartridge was subsequently washed with 0.5 ml of 0.1 mol/l HCl. After drying under vacuum for 30 s, the first elution was carried out with 1 ml of acetonitrile. The cartridge was dried again under vacuum for 30 s, followed by a second elution with 1 ml of acetonitrile–25% aqueous ammonia (19:1, v/v). After 10 ml of glycerol:water (1:1, v/v) was added to each fraction, the eluates were evaporated separately under a gentle stream of nitrogen at 45 8C, giving glycerol droplets. The residues were redissolved in 50 ml of water:methanol (9:1 for the first fraction, 1:9 for the second fraction, v/v), followed by membrane filtration through Ultrafree MC filters. The filtrates were transferred to plastic vials, and a pair of the extracts was subject to the LC/MS analyses separately using the corresponding acquisition programs. Twenty microliters of each was injected via the built-in autosampler. 2.6. Extraction recovery of Oasis MCX cartridge Drug concentrations for recovery tests were 500 ng/ml (A) and 50 ng/ml (B) in serum (n = 5). In order to avoid peak overlapping especially between amobarbital and pentobarbital, the analytes were divided into two groups and

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Table 2 Filter adsorption, extraction recoveries and detection limits Type

Analyte

Filtration recoveries (%, n = 2)

Monitor wavelength (nm) A A A A A A A A

Amobarbital Apronalide Barbital Bromisovalum Pentobarbital Phenobarbital Secobarbital Trichloroacetic acid

216 240 213 232 216 240 216 211

B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B

Alprazolam Bromazepam Brotizolam Chlordiazepoxide Chlorpromazine Clonazepam Clotiazepam Delorazepam N-Desmethyldiazepam N-Desmethylfludiazepam Diazepam Diphenhydramine Estazolam Etizolam Fludiazepam Flunitrazepam Flutazolam Haloxazolam No. 574 Hydroxyzine Lorazepam Lormetazepam Midazolam Nimetazepam Nitrazepam Promethazine Quazepam Rilmazafone M-4 Tandospirone Triazolam Zolpidem Zopiclone

226 237 243 263 256 308 244 232 232 232 232 m/z 256 (MS) 232 244 234 308 246 232 m/z 375 (MS) 232 233 232 262 260 253 287 234 244 227 244 305

Dissolved in 10% methanol

Extraction recoveries (%, n = 5) Dissolved in 90% methanol

Recovery

Detection limits (ng/ml)

S.D.

94.9 97.2 96.2 99.6 99.6 99.5 94.5 99.3

98.3 98.8 98.4 – 99.1 – 98.0 –

100.5 99.5 99.0 101.2 87.4 92.4 102.2 81.3

4.8 6.1 6.4 4.8 4.1 7.0 4.9 2.4

97.2 98.0 95.3 80.4 7.6 91.6 79.3 93.0 85.7 89.3 85.4 19.2 97.2 89.0 90.3 85.3 62.6 93.0 6.4 96.9 93.8 72.9 91.5 96.3 2.7 23.7 100.8 73.8 92.0 61.0 73.1

100.3 101.5 94.7 99.3 36.2 104.4 94.6 100.2 103.8 98.8 99.7 78.5 100.3 103.4 96.2 98.0 101.9 99.1 94.1 100.0 98.6 98.2 95.0 100.3 78.6 100.0 97.0 77.1 102.2 90.5 43.4

91.5 97.7 96.0 107.7 82.3 80.4 86.6 97.1 87.0 96.6 96.6 83.2 93.5 91.2 90.8 94.3 83.8 88.0 93.4 81.9 45.0 83.3 86.3 89.1 94.6 61.1 36.9 91.1 90.6 87.9 90.0

2.0 1.6 3.2 3.0 2.4 4.9 2.1 6.6 4.3 3.1 4.3 2.9 1.6 7.7 3.4 3.6 3.1 6.5 1.2 4.2 13.4 3.5 5.0 2.8 3.6 25.3 6.4 2.8 4.5 3.7 2.1

Serum d

Serum e

Serum f

1 1 10 10 1 5 1 50

1 1 10 10 1 5 1 50

5 5 10 10 5 5 5 50

0.5 1 0.5 1 1 1 0.1 0.5 0.5 0.1 0.1 0.5 0.5 0.1 0.1 0.5 1 0.5 1 5 5 0.1 0.5 0.5 1 5 1 0.1 0.5 0.1 5

0.5 1 0.5 1 1 1 0.5 0.5 0.5 0.1 0.1 0.5 0.5 0.1 0.1 0.5 1 0.5 1 5 5 0.1 0.5 0.5 1 5 1 0.1 0.5 0.1 5

0.5 1 0.5 0.5 1 1 0.1 0.5 0.5 0.1 0.1 0.5 0.5 0.1 0.1 0.5 1 0.5 1 5 5 0.1 0.1 0.5 1 5 1 0.1 0.5 0.1 5

–, not determined due to peak distortion; S.D., standard deviation.

investigated by LC/MS separately. To compensate for the volume variation of the final LC/MS samples, each extract was dissolved in 50 ml of water:methanol (9:1 for the first fraction, 1:9 for the second fraction, v/v) containing 250 ng/ ml ethenzamide and the calculations were performed by the peak–area ratios to ethenzamide. The monitoring ion and cone voltage for ethenzamide were m/z 166 V and 30 V, respectively, and the retention time of ethenzamide almost corresponded to that of phenobarbital. When preparing

reference samples, one of the two standard mixtures was added to each fraction of a blank serum extract, and the average (n = 3) was considered as 100% value of the recovery. 2.7. Detection limits The following concentrations (n = 2) were employed for determining the detection limits: A, 50, 10, 5, 1 (ng/ml);

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Fig. 1. Metabolic pathways for sedative-hypnotic drugs in circulating blood.

B, 5, 1, 0.5, 0.1 (ng/ml). Analytes were divided into two groups as mentioned above. Three human sera (d–f) were used as matrices. The concentration required to produce a signal-to-noise ratio of better than 5:1 (determined by the peak-to-peak method) was accepted as the detection limits.

2.8. Matrix effects Six human sera (a–f), obtained commercially, were submitted to extraction as described above to evaluate the existence of the endogenous interferences that may cause

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Fig. 2. Infusion experiment. For the blank run, water:methanol (9:1, v/v) was injected into the column instead of a serum extract. Cone voltages used for the monitoring at m/z 117 and 163 were 30 V and 20 V, respectively. RT, retention time; TCA, trichloroacetic acid.

false positive. By using the same extracts of sera d–f, postcolumn infusion experiments [16] were carried out. Illustration of the experiments is shown in Fig. 2 (upper). Chromatographic separations for the serum extracts were carried out while trichloroacetic acid (TCA, 20 mg/ml) in water:methanol (1:1, v/v) was continuously infused postcolumn through a tee at a flow rate of 2 ml/min. At the same time, negative ESI signals of TCA (scan mode, cone voltage 20 V and 30 V) were observed.

3. Results 3.1. LC/MS conditions Mass chromatograms of the extracts obtained from the spiked serum are shown in Fig. 3. Satisfactory separation was

achieved for most of the analytes by employing relatively long gradient cycle (80 min), although careful choices of the monitored ions were necessary for resolving overlapped peaks, as described below. Stability of the retention times was generally excellent, but retention times of diphenhydramine, chlorpromazine and promethazine were liable to shift by the concentration of the analytes and condition of the column. For the maximum MS response, the optimum polarity and cone voltages were examined for each analyte, and these values were employed for the settings of selected ion monitoring (SIM), along with the fractionation and retention time of the analytes (Table 1). Some minor isotopes were monitored to resolve overlapping peaks. For example, the second isotopic peak at m/z 311 in addition to the first at m/z 309 was employed for the detection of alprazolam because the peak at m/z 309 overlapped with that for zolpidem (Fig. 3). Similarly, the

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Fig. 3. Mass chromatograms of serum extracts. Serum d was used for the matrix. Non-basic analytes (A (except for amobarbital), 1000 ng/ml in serum) and basic analytes (B, 100 ng/ml in serum) were extracted in the first fraction (upper) and the second fraction (lower), respectively. RT, retention time; n, negative mode; TCA, trichloroacetic acid.

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3.2. Sample preparation

Fig. 4. Mass spectra of lormetazepam and haloxazolam metabolite No. 574. Cone voltages were set to 30 V (lormetazepam) and 50 V (the haloxazolam metabolite).

retention time and the base mass peak for lormetazepam were consistent with those of the haloxazolam metabolite (Fig. 3), but it was possible to resolve them by comparing the spectral patterns (lormetazepam, m/z 335 and 337; the haloxazolam metabolite, m/z 333, 335 and a trace of 337). The full-scan MS spectra of these two compounds are shown in Fig. 4. Amobarbital could not be discriminated from pentobarbital by this method because the retention times and mass spectra were completely identical.

In order to minimize loss of analytes by evaporation, glycerol was added prior to the evaporation process. The addition of glycerol improved the recovery of TCA; nevertheless, excess evaporation impaired the recovery (data not shown). Adsorption losses of the analytes on PVDF membranes were evaluated (Table 2). An extract is usually reconstituted in the initial mobile phase, but dissolving in 10% methanol, equal to the initial mobile phase of this method, resulted in the considerable losses of some basic analytes such as chlorpromazine, diphenhydramine, hydroxyzine, promethazine and quazepam on filtration. Dissolving the second fraction (B) in 90% methanol, which had no effect on retention times and peak shapes, dramatically improved the adsorption losses of hydrophobic analytes on filtration. Although recovery of chlorpromazine was still low (36.2%) and recovery of zopiclone was getting low (43.4%), water: methanol (1:9) was employed for the reconstitution of the second fraction. Extraction efficiencies are shown in Table 2. All of the analytes were extracted in the expected order and the extraction efficiencies were sufficient for screening although some of the recoveries were relatively low (i.e., lormetaze-

Fig. 5. Comparison of the mass chromatograms of bromisovalum extracted from different sera. RT, retention time.

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pam, quazepam and rilmazafone M-4). Most benzodiazepines were eluted into the second fraction only, but lormetazepam was eluted into the first fraction as well as the second fraction (data not shown).

3.3. Detection limits Detection limits in serum were determined (Table 2). Three sera, derived from different donors, were used for the

Fig. 6. Mass chromatograms derived from a real-case sample. The order of chromatograms are consistent with Fig. 3. Peaks #1–3 correspond to bromisovalum, apronalide and diphenhydramine, respectively.

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matrices. The non-basic analytes were detectable at concentrations above 1–10 ng/ml in serum except for TCA (50 ng/ml), and the basic analytes were detectable at concentrations above 0.1–1 ng/ml in serum except for lorazepam, lormetazepam, quazepam and zopiclone (5 ng/ml). There was substantially no difference in the detection limits between sera. 3.4. Matrix effects By monitoring isotopic ions and fragment ions in addition to the major protonated molecules, interferences were discriminated from the analytes. Examples of bromisovalum (50 ng/ml in sera d–f) are shown in Fig. 5. The retention time of bromisovalum was 26.0 min. The peaks at m/z 223 and 225 are derived from the protonated molecules, and the peaks at m/z 180 and 182 are derived from the fragment ions of bromisovalum. Large interferences were observed near the peaks of bromisovalum at m/z 223 and 182; on the other hand, the peaks of bromisovalum at m/z 225 and 180 were free from interference. Any interference that was unable to discriminate from the analytes (at concentrations higher than the detection limits) was not observed in six blank matrices. The postcolumn infusion experiment resulted in Fig. 2. Severe ion suppression and interferences were observed in the early period of the chromatography cycle, but at the retention time of TCA, no matrix effect that would impair the signal intensity was observed. Similarly, barbital (ESI negative, cone voltage 30 V) and bromisovalum (ESI positive, cone voltage 30 V and 60 V) were tested, and no suppression was observed at the retention time of the corresponding drug (data not shown). 3.5. Forensic application The present method was applied to a case of suicide by burning charcoal in a small space. Empty packages of nonprescription medicines containing bromisovalum, apronalide and/or diphenhydramine were found at the scene. This screening resulted in the detection of these three compounds (peaks #1–3 in Fig. 6), and the result was verified by GC/MS analyses (data not shown).

4. Discussion 4.1. Choice of analytes based on pharmacokinetics In general, drugs persist in circulating blood for at least several hours after an oral administration, but some types of sedative-hypnotics are quickly eliminated from the blood. Therefore, in order to demonstrate the use of the target drugs, analytes were chosen on the basis of pharmacokinetic data. The metabolism of sedative-hypnotic drugs is summarized in Table 1 and Fig. 1.

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Chloral hydrate and triclofos are immediately biotransformed to 2,2,2-trichloroethanol, the active metabolite, followed by oxidation to TCA (Fig. 1a). It has been reported that chloral hydrate is not detectable in plasma after a therapeutic dose, but that TCA levels are higher than 5 mg/ml, 100-fold higher than the detection limit of this method, even after 192 h [17]. A similar mechanism has been proposed for the metabolism of triclofos [18]. Therefore, TCA would be the most suitable alternative for the demonstration of the two chlorinated drugs after their ingestion (Table 1), although TCA is pharmacologically inactive. Some benzodiazepine derivatives contain metabolically unstable moieties that are easily removed. Oxazolam, cloxazolam, haloxazolam, mexazolam and flutazolam are benzodiazepine-related compounds that have oxazole rings, which are transformed to the corresponding benzodiazepines (Fig. 1b–e). It has been reported that the parent drugs in blood are undetectable (cloxazolam [19], haloxazolam [20] and mexazolam [21]) or present in trace amounts in some individuals (oxazolam [22]). Therefore, not the precursors but the parent benzodiazepines were employed as the target analyte (Table 1). In the case of flutazolam, it has been reported that the parent drug, as well as both the corresponding benzodiazepine and its N-desalkyl form (N-desmethylfludiazepam), can be observed in serum, and the level of Ndesmethylfludiazepam in serum remained nearly constant (about 5 ng/ml) for a day, whereas those of the parent drug and the corresponding benzodiazepine decreased over time [23]. Thus, monitoring of both the parent drug and Ndesmethylfludiazepam is preferable in terms of demonstrating flutazolam use. Rilmazafone is a ring-opened benzodiazepine-derivative that is a precursor of several active metabolites. After the degradation of the glycine moiety of rilmazafone, it spontaneously forms a ring-closed benzodiazepine, followed by demethylation and oxidation of the lateral N-dimethylamide group [24] (Fig. 1f). It has been reported that the resulting carbonate (M-4) is inactive but is the major metabolite in plasma for periods of up to 6 h, while the parent compound was not observed after therapeutic dosing [24]. Although an analytical standard of rilmazafone M-4 is not available commercially, the complete hydrolysis of rilmazafone by refluxing in 3 mol/l HCl gave M-4 as a white precipitate (data not shown). The 1,4-benzodiazepines that have halogen atoms at certain positions (i.e., 7-Cl and 20 -F: ethyl loflazepate, fludiazepam, flurazepam, flutazolam, flutoprazepam and quazepam) are all metabolized to N-desmethylfludiazepam, an active metabolite (Fig. 1b). Ethyl loflazepate, flurazepam, flutazolam and flutoprazepam are immediately metabolized to N-desmethylfludiazepam [23,25–27]. On the other hand, a certain amount of fludiazepam and quazepam appears in circulating blood after the administration of a therapeutic dose [28,29]. Therefore, N-desmethylfludiazepam, fludiazepam and quazepam were chosen for analytes.

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N-Desmethyldiazepam (nordiazepam, nordazepam) is also a common metabolite of certain benzodiazepines such as diazepam [30], prazepam [30,31], oxazolam [22,31], clorazepate [30,31], medazepam (via diazepam) [32] and chlordiazepoxide [33] (Fig. 1c). Medazepam was not monitored in this method, since it has been reported that N-desmethyldiazepam is maintained at a higher level (ca. 50–100 ng/ml) for several days after the oral administration of medazepam, while the parent drug was observed only in the early period and the level of diazepam was maintained at a lower constant value (ca. 10 ng/ml), although considerable differences between individuals were noted [32]. The appearance of the other drugs in circulating blood, not mentioned above, was confirmed by pharmacokinetics data found in the literatures and package inserts of the corresponding pharmaceutical products. The detection limits for the analytes were acceptable, although those of lorazepam, lormetazepam, quazepam and zopiclone (5 ng/ml) were inferior to other basic analytes. Lormetazepam has the lowest maximum blood concentration (Cmax) (12.4 ng/ml) among these four drugs, but elimination of lormetazepam from circulating blood is relatively slow (half life, 13.6 h) [34]. Therefore, the detection limit estimated for lormetazepam is sufficient to demonstrate its intake for about half a day after ingestion. Because the present method focuses on screening, additional analyses such as GC/MS and LC/MS/MS are thought to be essential to achieve definite identifications. Moreover, because some of the analytes can be derived from two or more drugs, the additional analyses are also required to specify the ingested drug. It should be kept in mind that a parent drug itself, such as diazepam, lorazepam, nitrazepam, pentobarbital and phenobarbital, could be a metabolite of others, as shown in Fig. 1 [32,34–37]. 4.2. LC/MS conditions In comparison with tandem LC/MS/MS measurements, a single LC/MS measurement requires finer chromatographic separation to discriminate possible drugs from a large number of compounds that consist, not only of analytes, but also of numerous interferences in biological matrices as well, that is, high-throughput chromatography may not be appropriate when single LC/MS is employed. SIM enables a cone voltage at the optimum value to be set which would provide the best sensitivity for each analyte, and of course, SIM is essentially more sensitive than fullscan mode in a quadrupole mass analyzer. On the other hand, spectral information is not available with SIM. To compensate the drawback of SIM, some minor isotopic ions and some fragment ions were also monitored in this method, which was useful for the discrimination of the analytes from interferences (Fig. 5). Fragmentation was introduced only for some interference-rich analytes because an increase in the number of simultaneous acquisitions decreases the number of data points per peak.

4.3. Sample preparation Sample filtration prior to injection is important because presence of particulates in the samples can reduce column life. On the other hand, there is a possibility of adsorption on the filter membrane. Using a methanol-rich solvent effectively reduced adsorption of the hydrophobic analytes, such as promethazine and hydroxyzine, on the fluoropolymer filter membrane, although the recovery of zopiclone was unexpectedly deteriorated. The first fraction contained more endogenous interferences than the second fraction for two reasons: there is considerable non-basic interference, and only aqueous washing was carried out before the first elution. A further washing step was not employed because barbital and TCAwere eluted, even with 5% methanol (data not shown). However, this is not a serious problem because therapeutic levels of non-basic sedative-hypnotics in blood are higher than those of basic drugs, and ion suppression effects caused by the interferences in the first fraction had been estimated in this study. 4.4. Matrix effects Ion suppression caused by coeluted compounds is an inevitable phenomenon for electrospray ionization. This can deteriorate the response of the spectrometer, and consequently, the detection limits. In the present method, polar analytes that are barely retained by the analytical column, such as TCA, are obviously at the high risk of ion suppression. Accordingly, infusion experiments of TCA, barbital and bromisovalum were carried out for evaluating the interferences that cause ion suppression. Moreover, the detection limits of all the analytes were determined using three individual sera as the matrices. These experiments demonstrated that there is no evidence of ion suppression. In conclusion, the present method will be useful for preliminary screening of sedative-hypnotics drugs in serum. From practical point of view, it will be beneficial to include other drugs that cause sedation such as g-hydroxybutyrate, classic antidepressants, reserpine and opiates. Further studies on the applicability to real-case samples are now under investigation. Acknowledgements We are grateful to Dr. Hitoshi Sekine (Saitama prefectural police H.Q.) for providing drug standards. We also thank Dr. Shinichi Suzuki (National Research Institute of Police Science) for valuable information on drug metabolism. References [1] O.H. Drummer, Methods for the measurement of benzodiazepines in biological samples, J. Chromatogr. B 713 (1998) 201–225.

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